Association of tuberculosis risk with genetic polymorphisms of the immune checkpoint genes PDCD1, CTLA-4, and TIM3

The immune checkpoint proteins were reported to involve to host resistance to Mycobacteria tuberculosis (Mtb). Here, we evaluated 11 single nucleotide polymorphisms (SNPs) in PDCD1, CTLA4, and HAVCR2 genes between participants with and without TB infection. Genomic DNA isolated from 285 patients with TB and 270 controls without TB infection were used to perform the genotyping assay. Odds ratios were used to characterize the association of 11 SNPs with TB risk. In this study, the various genotypes of the 11 SNPs did not differ significantly in frequency between the non-TB and TB groups. When patients were stratified by sex, however, men differed significantly from women in genotype frequencies at HAVCR2 rs13170556. Odds ratios indicated that rs2227982, rs13170556, rs231775, and rs231779 were sex-specifically associated with TB risk. In addition, the combinations of rs2227982/rs13170556 GA/TC in men and the A-C-C haplotype of rs231775-rs231777-rs231779 in women were significantly associated with TB risk. Our results indicate that rs2227982 in PDCD1 and rs13170556 in HAVCR2 are associated with increased TB susceptibility in men and that the CTLA4 haplotype appears protective against TB in women.


Introduction
Tuberculosis (TB) is an ancient disease primarily caused by Mycobacterium tuberculosis (Mtb).It remains a socially significant condition and was the thirteenth leading cause of death in 2019 [1].In recent years, associations of genetic variations in host immune-related genes and TB susceptibility have been reported [2][3][4], and some research has implicated immune checkpoint proteins in TB risk [5,6].These proteins, including programmed cell death-1 (PD-1), cytotoxic T-lymphocyte antigen-4 (CTLA-4), and T-cell immunoglobulin and mucin domain 3 (TIM3), represent the immune regulatory function of co-inhibitory receptors associated with T-cell dysfunction and exhaustion in many cancers [6][7][8].Although the possible involvement of immune checkpoint proteins in Mtb infection has attracted increasing focus [9][10][11], the association of their genetic polymorphisms with TB susceptibility is not well understood.Careful examination of the relationship between these polymorphisms and TB risk would increase understanding about human susceptibility to TB.
PD-1 that is encoded by the PDCD1 gene on human chromosome 2, which consists of 5 exons, is expressed during T-cell activation.Through its ligands-programmed cell death 1 ligand 1 (PD-L1) and PD-L2 it counters positive signals through the T-cell receptor and CD28 [12].Patients with active TB have higher levels of CD4+ T-cell PD-1 expression compared with unaffected controls [13,14].In addition, PD-1 −/-mice are more susceptible to Mtbinduced mortality because of excessive inflammation and uncontrolled bacterial proliferation in the lungs [15].Also in mice, PD-1 ligation decreases interferon (IFN)-γ production by CD4 + T cells and results in exacerbated pulmonary Mtb infection and early host mortality [16].CD4+ T cells and macrophages enhance phagocytosis and intracellular killing of Mtb in vitro though blocking the PD-1/PD-L1 pathway, suggesting an important role for PD-1 in Mtb infections [13].
CTLA4 is encoded by the CTLA4 gene that is located on human chromosome 2 and consists of 4 exons.CTLA-4, a transmembrane homodimer glycoprotein, is expressed by activated effector T cells and negatively regulates T-cell immune responses [17].Furthermore, patients with latent TB infection with Mtb have higher levels of PD-1, CTLA-4, and TIM3 in lymphocytes compared with unaffected controls, possibly because of an evasion of immune exhaustion in the Mtb infection [18].In peripheral blood mononuclear cells from patients with latent TB infection, Mtb culture-filtrate antigen was shown to induce production of IFN-γ and interleukin (IL)-17 after CTLA-4 blockade [19].
TIM3, a member of the TIM family of immunoregulatory proteins, is associated with regulation of immune responses in autoimmunity and cancer [20].TIM3 is encoded by the HAVCR2 gene on human chromosome 5, which consists of 7 exons.Previous research has indicated increased TIM3 expression in CD8+ T cells in mice during chronic Mtb infection [21,22].In patients with Mtb infection, high levels of TIM3 expression are also found in CD8+ T cells [23,24].Work with mouse models has shown that TIM3 influences T-cell exhaustion by decreasing production of IFN-γ and tumor necrosis factor [11].In chronically infected susceptible mice, blockade of TIM3 restores T-cell function and improves bacterial control [11].Taken together, these findings suggest an important role for PD-1, CTLA-4, and TIM3 in the host immune response during Mtb infection.Despite their involvement, however, the interaction of genetic polymorphisms of PDCD1, CTLA4, and HAVCR2 in TB susceptibility is not well known.
Associations of TB susceptibility with some single nucleotide polymorphisms (SNPs) of PDCD1 and CTLA4 have been reported [25][26][27].In PDCD1, rs7568402 is linked to increased TB risk [25], and rs3087243 AA in CTLA4 is negatively associated with severe pathology in pulmonary TB [26].In people of Southern Han Chinese ethnicity, rs231775 AG in CTLA4 has been associated with reduced risk for TB infection [27].However, whether other SNPs in PDCD1, CTLA4, and HAVCR2 are associated with TB risk has not been demonstrated.To address this gap, we investigated the association of 11 SNPs of PDCD1, CTLA4, and HAVCR2 genes with TB risk.

Study population
A total of 285 individuals diagnosed with Mtb infection were included in the study conducted at General Taoyuan Hospital in Taoyuan, Taiwan.We gathered medical record information for outpatients from January 2022 to December 2022.The inclusion criteria were age �20 years and diagnosis with active TB disease (i.e., evident TB lesions on X-ray or computed tomography, or positive results of sputum smears and cultures for Mtb).As a control group, 270 adult participants without a history of TB disease were enrolled.To avoid the interference of some antiviral and anti-cancer drugs, patients with cancer or other immune-related diseases and viral infections (e.g., hepatitis B, hepatitis C, HIV) were excluded.The study protocol was developed according to the ethical guidelines of the 1975 Declaration of Helsinki and was approved by the Ethics Committee of Taoyuan General Hospital, Taoyuan, Taiwan (TYGH110033).All participants provided written informed consent for this study.

DNA extraction and SNP selection
Genomic DNA was extracted from peripheral blood cells or oral swabs collected from the enrolled participants respectively using a Quick-DNA™ Miniprep Kit or a QIAamp DNA Mini Kit according to the manufacturer's instructions.Briefly, cell pellets from 300 μl of whole blood or buccal swab were lysed using the respective lysis buffer.After centrifugation at 14,000 ×g for 1 min, the supernatant was applied to the respective spin column for DNA purification and washed twice with wash buffer.After sterile distilled deionized water (150 μl) was added in the spin column for a 2-min incubation at room temperature, DNA was eluted by centrifuging at 14,000 ×g for 2 min.The extracted genomic DNA was analyzed using agarose gel electrophoresis, quantified by spectrophotometry, and stored at -80˚C until SNP genotyping (Feng Chi Biotech Corp., Taipei, Taiwan).The tSNPs within the genomic regions of PDCD1, CTLA4, and HAVCR2, along with the 1500 base pairs upstream, were chosen using the SeattleSNPs website (http://pga.mbt.washington.edu/education.html)based on data specific to the Han Chinese population in Beijing (HapMap-HCB).The SeattleSNPs database revealed a limited number of polymorphisms (MAF > 0) within our designated region, with four SNPs identified in PDCD1 and three in CTLA4.In the HAVCR2 region, numerous SNPs were documented and organized into four bins.From these, we opted for four SNPs in PDCD1, three in CTLA4, and four in HAVCR2 (one from each bin) for subsequent genotyping.The characteristics of the selected SNPs were presented in S1 Table.

Genotyping assay
Genotyping of the tag SNPs was performed using the Agena MassARRAY platform with iPLEX reagent chemistry (Agena, San Diego, CA).Briefly, after amplification by PCR of the genomic DNA regions containing the tag SNPs, a single base extension reaction was performed to generate allele-specific diagnostic products.These products were identified by their unique molecular weights, using matrix-assisted laser desorption ionization-time-of-flight mass spectrometry.After allele-specific diagnostic products were loaded onto a matrix pad of a Spectro-CHIP (Agena), the Spectro-CHIP was analyzed using a MassARRAY Analyzer 4, and results were evaluated using clustering analysis with TYPER 4.0 software.The specific primer sequences of PCR and unextended primers used in this study are shown in S2 Table.

Statistical analysis
The intermarker linkage disequilibrium (LD) of tag SNPs in the three genes was estimated, and haplotype blocks were defined based on r 2 and D' values, using Haploview v.4.2.Unpaired Student's t-test was used to examine differences in age between non-TB and TB groups.Logistic regression and χ 2 tests were used for association analyses, and odds ratios (ORs) and 95% confidence intervals (CIs) for TB risk were calculated.All statistical assessments were carried out using SPSS 21.0 software (SPSS Inc., Chicago, IL, USA).

Participant characteristics
A total of 555 people (430 men and 125 women) were enrolled.As indicated in Table 1, we found no significant difference in sex distribution between the two groups (p = 0.166, χ 2 ).The mean age of participants with TB was 57 years (range 20-92 years), and the mean age of unaffected controls was 71 years (range 22-99 years).In the overall population and within the sexdefined subgroups, the TB and non-TB groups differed significantly in age (t-tests).

Association of PDCD1, CTLA4, and HAVCR2 polymorphisms with TB
When the 11 SNPs in PDCD1, CTLA4, and HAVCR2 were genotyped, their genotype frequency distributions were consistent with Hardy-Weinberg equilibrium (Table 2).In the LD plot, one haploblock was identified at PDCD1 and another at CTLA4 (Fig 1).Selected SNPs did not differ significantly in genotype frequencies, and TB risk was not associated with these frequencies in the comparison of the non-TB and TB groups in the total population (Table 2).
Logistic regression to evaluate interactions of sex and genotype yielded a significant interaction of sex and the rs13170556 genotype in HAVCR2 (S3 Table ).In addition, genotype and age (<65 vs �65 years) showed a significant interaction (S3 Table ).
In age-stratified groups, genotype frequencies of the 11 SNPs did not differ significantly between those <65 years and those �65 years (S4 and S5 Tables).In addition, in these two groups, SNPs were not significantly associated with TB risk (S4 and S5 Tables).

Association of haplotypes of PDCD1 and CTLA4 with TB risk
The results of LD analysis for polymorphisms of PDCD1, CTLA4, and HAVCR2 are presented in Fig 1 .Of the eight possible haplotypes, six haplotypes of PDCD1 rs2227982-rs7421861-rs6710479 and four haplotypes of CTLA4 rs231775-rs231777-rs231779   were detected (Table 6).In comparison with the most common haplotype of PDCD1 (A-A-T haplotype of rs2227982-rs7421861-rs6710479) or CTLA4 (G-C-T haplotype of rs231775-rs231777-rs231779), no haplotype was significantly associated with TB risk overall, among male participants, or in the age-stratified populations (Table 6 and S6 Table ).Female participants with the A-C-C haplotype of CTLA4 had significantly lower risk of TB (aOR = 0.488, 95% CI = 0.263-0.905,p = 0.023) compared with those who had the most common haplotype (G-C-T) of rs231775-rs231777-rs231779 (Table 6).

Discussion
Previous groups have reported that some polymorphisms of PDCD1, CTLA4, and HAVCR2 are associated with TB risk [25][26][27][28].In this study, we found a sex-dependent association of TB risk with the AG genotype of PDCD1 rs2227982, TC genotype of HAVCR2 rs13170556, AG genotype of CTLA4 rs231775, and CT genotype of CTLA4 rs231779.In men, the AG/TC combination of PDCD1/HAVCR2 was significantly associated with TB risk.In women, our  haplotype analysis showed that the A-C-C haplotype of CTLA4 rs231775-rs231777-rs231779 was associated with reduced susceptibility to TB.Our findings imply that genetic polymorphisms of PDCD1, CTLA4, and HAVCR2 have an important role in TB susceptibility.
In men, OR analysis showed a significant association of rs2227982 AG with increased risk of TB under the additive model.PDCD1 is located on chromosome 2, and rs2227982 (+7625 C>T) lies in the fifth exon of PDCD1.The rs2227982 base substitution can lead to an exchange of valine for alanine, possibly influencing the activity of PD-1 [29].In the GTExPortal database, rs2227982 is reported to be associated with PDCD1 expression (according to expression quantitative trait loci analysis) in subcutaneous and visceral adipose tissues (https://gtexportal. org/home/snp/rs2227982) [30].In addition, previous studies have indicated associations of PDCD1 rs2227982 with host susceptibility to hepatitis B infection and risk of Mycobacterium avium complex lung disease in women [31,32].A significant relationship between serum soluble PD-1 levels and different rs2227982 genotypes also has been reported [32].PD-1 regulates Mtb infection and rs2227982 is associated with PD-1 expression, which may explain the association of rs2227982 with TB risk.
The distribution analyses in the current work revealed a higher frequency in the male population of the rs13170556 TC genotype among patients compared with healthy controls; thus, this genotype may play a predisposing role in TB infection.Human HAVCR2 is located on chromosome 5, and the SNP rs13170556 lies in the fourth intron of HAVCR2.The OR analysis showed a sex-dependent association of the rs13170556 TC genotype with TB risk.Sex-dependent associations of rs4331426 and rs35037722 with TB risk have been reported previously, similar to studies in the Han Taiwanese population [3,33].The GTExPortal database indicates an association of rs13170556 with HAVCR2 expression in testis tissue (https://gtexportal.org/ home/snp/rs13170556) [30].In addition, HAVCR2 genetic variations have been linked to increased risk of osteoarthritis, possibly because of upregulation of IFN-γ expression by CD4 + T cells [34].Based on these findings, we hypothesize that rs13170556 may regulate TIM3 and INF-γ expression, in turn influencing susceptibility to TB.
We also found that rs231775 AG and rs231779 CT are associated with reduced risk of TB, similar to research showing that rs231775 AG is a protective factor against TB in a Southern Han Chinese population [27].In addition, we found that the A-C-C haplotype of the LD block (rs231775-rs231777-rs231779) in CTLA4 is associated with reduced risk for TB in women.Some previous research has linked this LD block in CTLA4 with immune-related diseases, such as Graves' disease and Rasmussen syndrome [35,36].Human CTLA4 is located on chromosome 2, and rs231775 (+49A>G) and rs231779 are respectively located in the first exon and intron of the CTLA4 gene.The rs231775 base substitution at position 17 in exon 1 of CTLA4 can lead to a change from threonine to alanine [37].In testis tissue, the association of rs231775 and rs231779 with CTLA4 expression has been reported in the GTExPortal database (https://gtexportal.org/home/snp/rs231775; https://gtexportal.org/home/snp/rs231779)[30].
In addition, other studies have indicated an association of rs231775 with CTLA-4 expression and an effect on the affinity of CTLA-4 for B7-1 (CD80) in many cancers [38,39].CTLA4 polymorphisms also have been associated with increased secretion of cytokines such as IFN-γ and IL-2 and an enhanced immune response [40].Taken together, the findings regarding rs231775 and rs231779 suggest an influence on susceptibility to TB through regulation of CTLA-4 expression and T-lymphocyte response.
The important effects of several host genetic factors on tuberculosis infection have been reported [41].PD-1, CTLA4, and TIM3 were associated with host immune response against Mtb infection [6].In this study, we indicated the associations of PDCD1 rs2227982, HAVCR2 rs13170556, CTLA4 rs231775, and CTLA4 rs231779 with TB risk.Though the number of subjects with different TB severity (cavitation and pleural effusion) is not sufficient to evaluate the association of selected SNPs with TB severity in our study, some previous studies have reported the association of these SNPs with clinical outcome of diseases, such as TB, primary biliary cirrhosis, and Hepatitis C [26,[42][43][44].In PDCD1 rs2227982, TB patients with TC or CC genotype have higher rate of tuberculous cavity than those with TT genotype [42].In previous study of African Population, the CTLA4 haplotype (rs11571315-rs733618-rs4553808-rs231774-rs231775-rs231777-rs3087243; G-A-A-A-G-C-A) of was associated with cavities in patients with TB [26].The CTLA4 haplotype (rs231775-rs231777-rs3087243-rs231725; G-C-G-A) was a protective factor for progression of primary biliary cirrhosis in Japanese patients [43].Though the association between HAVCR2 rs13170556 with clinical outcomes of TB is unknown, the protective effects of rs13170556 TC/ CC genotypes with the F protein on the outcomes of HCV infection was reported [44].In addition, the GTExPortal database indicates an association of PDCD1 rs2227982, HAVCR2 rs13170556, CTLA4 rs231775, and CTLA4 rs231779 with their gene expression in different tissues.Taken together, these finding may suggest that different genotypes in these SNPs may affect the host immune response against Mtb infection and clinical outcome of TB by regulating their gene expression.
The sex-dependent association of PDCD1, CTLA4, and HAVCR2 polymorphisms with TB susceptibility were found in our study.Some previous studies also indicated that PDCD1, CTLA4, and HAVCR2 polymorphisms were sex-dependently associated with MAC-LD risk, the resolution of hepatitis C virus infection, and the outcomes of HCV infection, respectively [32,44,45].In addition, previous study indicated that the effects of some sex-specific factors on the immune response and women have a higher prevalence of autoimmune diseases compared with men [46].The difference in function of CD4+CD25+ regulatory T-cells was observed in male and female mice [47].Taken together, these observations may suggest that the difference in sex-dependent effect of PDCD1, CTLA4, and HAVCR2 polymorphisms on immune response against Mtb infection.In our study, odds ratio analysis showed the significant associations of heterozygous genotype in PDCD1 rs2227982, HAVCR2 rs13170556, CTLA4 rs231775, and CTLA4 rs231779 with TB risk.In previous studies, the significant associations of heterozygous genotype of PDCD1 rs2227982 and HAVCR2 rs13170556 with the risk for breast cancer and the outcomes of HCV infection were reported, respectively [44,48].Though the transcriptional regulatory mechanism of different genotypes in these SNPs did not evaluate in our study, the differences in gene expression between heterozygous and homozygous genotypes of these SNPs in different tissues were found in the GTExPortal database [30].Based on these findings, we hypothesize that the difference in gene expression between heterozygous and homozygous genotypes of these SNPs may influence immune response against Mtb infection.However, our findings still need more researches with large sample sizes to validate.In future, the transcriptional regulatory mechanism of these SNPs and serum levels of soluble PD-1, CTLA4, and TIM3 should be investigated.
Our study has some limitations.First, stratification by sex yielded small group sizes that could have led to an underestimation of significance.Future studies should include a larger number of participants in each group to ensure sufficient statistical power.Second, because young participants rarely have clinical evidence for the exclusion criteria, it resulted in the significant difference in age of participants between TB and non-TB groups.Third, we did not evaluate some non-genetic factors affecting susceptibility to TB, such as host status (TB contact and latent TB infection) and environment (air pollution and tobacco smoke), and thus cannot exclude the possibility of some influence of these factors in the analyses.In future, to avoid the cofounder effects, OR analysis with adjustment for non-genetic factors of subjects should be performed.

Conclusions
We identified a significant difference in genotype frequencies of HAVCR2 rs13170556 between men with and without TB.Our OR analysis showed sex-dependent associations of TB susceptibility and AG heterozygosity at PDCD1 rs2227982, TC heterozygosity at HAVCR2 rs13170556, AG heterozygosity at CTLA4 rs231775, and CT heterozygosity at CTLA4 rs231779.In addition, the GA/TC combination of PDCD1 rs2227982/HAVCR2 rs13170556 was associated with increased TB risk in men, and the A-C-C haplotype at CTLA4 rs231775-rs231777-rs231779 was associated with reduced risk in women.Overall, PDCD1, CTLA4, and HAVCR2 polymorphisms are sex-dependently associated with susceptibility to TB.As the TB statistics of the Taiwan CDC has been showed that men have higher TB prevalence than women [49], our results may help explain the possibility of a difference in TB prevalence between non-TB and TB subjects in men and women.However, our findings still need more researches with large sample sizes in different ethnicities to validate.confidence interval; OR, odds ratio; Pc, the Bonferroni correction of P values.a χ 2 test.b Adj.= adjusted for age and sex by logistic regression.(DOCX)

Table 6 . Haplotype distribution of the two investigated PDCD1 and CTLA4 polymorphisms in the overall and sex-stratified populations.
Abbreviations: Ref., reference haplotype; CI, confidence interval; OR, odds ratio; Pc, the Bonferroni correction of P values.a χ 2 test b Adj.= adjusted for age and sex by logistic regression.https://doi.org/10.1371/journal.pone.0303431.t006